Saturday, 21 January 2012

Lag phase adapts bacteria to new environments

Forgotten to defrost the chicken overnight in the fridge? That’s fine—you can leave it to thaw at room temperature, right? It will be quicker, after all…

But, within just a few hours, the tiny bacteria hiding in frozen food such as chicken, beef or that left over Chinese takeaway can start to divide. Fail to cook the meal enough and you can end up with Salmonella food poisoning—an infection that affects around around 10,000 people in the UK each year (reported infections, at least) and is responsible for more than 100 deaths.

Salmonella bacteria lurking within food have to run a gauntlet of challenges in their attempt to infect the epithelial cells of our intestines. First, there’s stomach acid to content with, then there’s the normal intestinal flora preventing the attachment and growth of non-commensal species, and our immune system will do what it can to destroy any invading bacteria. For this reason, a large dose of bacteria has to be ingested for the infection to take hold—that’s around 106 cells in a healthy individual. Because Salmonella fails to be killed by freezing and can grow rapidly at 25⁰C, a relatively small number of microbes present in frozen food can multiply to dangerous levels if left at room temperature for long enough.

I’m fairly sure that, were I to be kept in a fridge, it would take me a while to start doing something if returned to room temperature. Salmonella also goes through this same period of adaptation to their new conditions and a recent study from the institute of Food Research in Norwich published in the Journal of Bacteriology looked into what is involved in Salmonella beginning to divide after a period of non-growth. Understanding what is happening in the pre-dividing bacteria could help to find new ways to prevent the growth of harmful bacteria in our food.

Like all bacteria, the growth of Salmonella can be divided into five phases as shown in the image below. The first stage, the lag phase, is a period of adjustment in which the bacteria prepares for active growth—the cells do not divide but, behind the scenes, they are actively preparing for growth. The cells need time to not only repair any damage accumulated during their period of non-growth, but they need to remake and switch on all the cellular machinery that was packed away while it wasn’t required. This is the least understood phase of bacterial growth, however, as there is little data to describe the processes occurring within a lag phase cell.

Growth phases of cultured bacteria. The lag phase involves adaptation to the new environment. This is followed by exponential doubling of the bacteria until they run out of space or nutrients and enter stationary phase. The culture may under a period of death until a long-term stationary phase is reached in which a small number of bacteria can survive for long periods of time in a non-growing state.

Once everything is ready, the cells enter exponential growth in which they double at a rate anywhere in between every 14 days for a slow-growing bacterium such as Mycobacterium leprae, which causes leprosy in humans, to every 20 minutes for Salmonella species. At this rate, one Salmonella bacterium could theoretically divide enough times to form a colony the size and mass of the Earth in less than one day. Of course, this doesn’t occur because the culture runs out of space and nutrients. Once things start to get crowded or food runs low, the culture enters stationary phase in which they hang around doing not very much, waiting for conditions to improve.

The fourth stage is a decline or death stage but, importantly, many bacteria are extremely tough and it can take a very long time for them to die. This means that a growth curve contains a final phase—long-term stationary phase. This particular phase of growth is interesting for research into a number of pathogens as it is thought to best represent the state in which bacteria survive during a number of diseases, such as in the latent stage of tuberculosis.

Move stationary phase bacteria to more favourable conditions and they are able to resume growth after a period of lag, and this is what happens with Salmonella. The problem with understanding exactly how bacteria restarts growth is hampered by the fact that, by definition, few cells are present during lag phase. The Journal of Bacteriology paper described how 750 ml cultures of Salmonella were incubated at 25 ⁰C without shaking (usually used in the lab to increase oxygen availability in the culture) to represent the conditions experienced by Salmonella in room temperature food.

The researchers used RNA isolated from these bacteria to determine which genes are switched on during lag phase. They found that a number of genes are induced within the first four minutes of lag phase and, among them, were a number of genes required for metal uptake and utilisation. Perhaps these metals are required for active growth and represent a limiting factor in whether the cells can regrow or not. A better understanding of the processes occurring during lag phase will result in new ways to prevent growth of bacteria such as Salmonella, leading to new ways to prevent diseases such as food poisoning. For example, perhaps inhibitors could be added to certain foods that prevent the vital metal uptake pathways from functioning.

Graphical representation of the changes in gene expression during lag phase - red represents genes that are switched on and blue are down-regulated.

In addition, the paper addressed an important question—are stationary phase bacterial cells already primed to switch on vital genes as soon as they find themselves in conditions conducive to growth? The rapid induction of genes in Salmonella would suggest that this is the case. However, the researchers determined that this is not true. Instead, those genes switched on within the first four minutes have very strong binding sites for the cell’s transcription machinery that means they can be turned on extremely quickly.

Model showing the major processes occurring during lag phase, exponential phase, and stationary phase. Each symbol represents groups of functionally related proteins and is colored according to the level of expression of the appropriate genes under each growth condition. Blue shows that the relevant genes are expressed at low levels, yellow shows genes expressed at medium levels, and red shows genes expressed at high levels in each growth phase.

The authors proposed a model to explain the processes are occurring in the various phases of Salmonella growth, shown above. They conclude the paper by saying that ‘It seems fitting that as Salmonella was the first bacterium in which lag phase was studied, it is now the first Gram-negative bacterium to be understood at the level of global gene expression during lag phase.’